This study was carried out to investigate the effects of treated protein supplements with tannin extracted from pistachio by-product (P-PB) on in vitro gas production using fistulated sheep. One portion of P-PB was mixed with four portions of water for 48 h. The extracted product was sprayed on soybean meal (SBM) or canola meal (CM) with an equal ratio (1:1 v/w) and dried in the shade. The experimental treatments included in this study were: 1) untreated soybean meal (USB), 2) soybean meal treated with tannin extract (SBTT), 3) untreated canola meal (UCM) and 4) canola meal treated with tannin extract (CMTT). Kinetics of gas production was fitted to an exponential model. After 96 h of incubation, the medium size of each syringe was used for determining ammonia N (NH3-N) concentration using distillation method. The results obtained from this study showed that spraying tannin extract on protein supplement increased the amount of tannin to 4.4, 3.13in CM or SBM, respectively. Although gas production rate, fraction b and fraction c decreased by treated protein supplements in comparison with untreated protein supplements, the effects were not significant (P>0.05). The effect of tannin extract on NH3-N was significant (P≤0.05). The highest and the lowest content of NH3 were for SBM with the lowest and CMTT with the highest content of tannin, respectively. Tannin from P-PB decreased organic matter digestibility (OMD), metabolizable energy (ME) and short chain fatty acid (SCFA) concentrations in treated protein supplements (P≤0.05). Untreated soybean meal and CMTT had the highest and the lowest content of OMD, ME and SCFA, respectively. Manuscript profile